Abstract 26,700 deaths from prostate cancer (CaP) are anticipated in 2017 in the US alone; in one large trial it was shown that up to 74% of deaths due to CaP occurred in patients with metastatic disease at diagnosis. These patients are treated with androgen deprivation therapy but invariably progress to castration-resistant cancer (CRPC) which has limited therapeutic options and none that extend median overall survival of over 5 months. In CaP, tumor cells express several highly prostate-specific surface proteins ideally suited for antibody (Ab) targeting such as prostate-specific membrane Ag (PSMA), against which high-affinity Abs have been raised, but with limited anti-tumor efficacy. Better efficacy has been demonstrated with T-cell redirecting approaches which include T-cell redirecting bispecific Abs (T-BsAbs) and chimeric antigen receptor T cells (CAR-T) that induce the body?s own T-cells to kill the tumor. However, there still remains the unsolved issue of strong T-cells activation leading to dose limiting toxic immune activation, including cytokine release syndrome (CRS). The overall goal of the proposed project is to develop a T-BsAb against PSMA that combines our innovations of (1) a novel, first-in-class ?PSMA heavy chain only Ab (UniAb) and (2) a unique activating anti-CD3 domain that induces killing with minimal cytokine secretion and preferential activation of effector over regulatory T cells. This molecule is expected to show high specificity, reduced toxicity and a widened therapeutic window. The heavy chain only structure of UniAbs facilitates multivalency, as demonstrated by our BCMAxBCMAxCD3 trivalent, bispecific Ab currently undergoing IND-enabling in vivo studies in multiple myeloma, and on track to enter the clinic in 2018. Using our proprietary UniAb producing rats (UniRats) together with our unique NGS-based bioinformatics pipeline (TeneoSeek), we have previously identified high affinity anti-PSMA (?PSMA) Ab families demonstrating PSMA binding and shown to mediate killing of prostate tumor cell lines in vitro and in vivo. In this proposed study we will perform diversity screening to identify ?PSMA UniAbs with optimal functional characteristics for the construction of a therapeutic molecule. From the previously-identified families, we will select the most suitable, highest affinity Ab leads using competitive binding experiments, diversity screening, and functional characterization (specific aim #1). Next, we will identify the leads with optimal safety and efficacy characteristics. The leads will be cloned into T-BsAb constructs together with Teneobio?s low-activating ?CD3 moiety and evaluated in vitro and in vivo models of CaP for killing specificity of PSMA-positive tumor cells, as well as off target activation (specific aim #2). Finally we will identify the leads with optimal safety and efficacy characteristics in ex vivo models of CaP, where the leads will be assessed for cytotoxicity, T-cell activation and cytokine release (specific aim #3). In future work, the lead molecule will be advanced into IND-enabling studies including rodent and Cynomolgus PK studies, tissue cross reactivity, and evaluation of anti-drug Abs in preparation for an IND filing and phase 1 clinical trials in CRPC patients.